The invention relates to pharmaceutical compositions of antimicrobial activity containing associates (complexes) of hyaluronic acid as well as a process for the preparation thereof.
The invention furthermore relates to the use of these hyaluronic acid associates (complexes) for the preparation of pharmaceutical compositions of antimicrobial activity and a method for the treatment of microbes-induced clinical pictures.
Now, it has been found that associates (complexes) of hyaluronic acid, i.e. zinc and cobalt hyaluronates showed antimicrobial activity and especially they have favorable antibacterial and fungicidal effects against aerobic and anaerobic microorganisms.
Associates of deprotonated hyaluronic acid with 3d metal ions of 4th period of the Periodic Table such as zinc and cobalt hyaluronates with a curative effect especially on crural ulcer, decubitus or the like are discussed in the Hungarian patent specification No. 203,372.
The macromolecule known as hyaluronic acid usually occurring in the form of its sodium salt, is a compound known for more than 50 years. It had first been described by Meyer and al [J. Biol. Chem. 107, 629 (1934); J. Biol.Chem. 114, 689 (1939)]. Meyer isolated the hyaluronic acid under acidic conditions. However, the carboxyl groups are dissociated at a physiological pH value; and the name of the polysaccharide is sodium hyaluronate if the environmental cation is sodium. The structure determination was performed by Weissman et al [J. Am. Chem. Soc. 76, 1753 (1954)]. The determination of environmental cation is not always simple therefore, as proposed by Balxc3xa1zs (The Biology of Hyaluronan 1989, John Wiley and Sons, Ciba Foundation Symposium, p. 143), the general name of the polysaccharide (both hyaluronic acid and sodium hyaluronate) is hyaluronan.
Hyaluronic acid is a highly viscous native glucosaminoglycan containing alternating xcex21-4 glucosamine moieties; its molecular weight is between 50000 and several millions. Hyaluronic acide is found in the connective tissues of all mammals: it occurs in higher concentration in the skin, vitreous body of eye, synovial fluid, umbilical cord as well as cartilaginous tissue. The recovery of hyaluronic acid is an old task, the separation and use of an ultrapure hyaluronic acid are described e.g. in the U.S. Pat. Nos. 4,141,973 and 4,303,676 and in the European patent specification No. 0,144,019.
A number of references relating to the connection of hyaluronic acid with the wound-healing are found in the literature. According to Toole and Gross [B. P. Toole xc3xa9s Gross: xe2x80x9cThe extracellular matrix of the regenerating newt limb: synthesis and removal of hyaluronate prior to differentiationxe2x80x9d, Dev. Biol. 25, 55-57 (1971)], hyaluronic acid as the main constituent of the extracellular matrix is responsible for the migration of various cell types. The above authors stated by experimental investigations that, during the wound-healing, the local concentration of hyaluronic acid increased whereby the cellular reactions required to the tissue regeneration were stimulated. In addition to the above, other experimental results also confirm that hyaluronic acid regulates the wound-healing physiological cellular events by providing the optimum conditions for migration and proliferation of all cells sharing in the tissue regeneration.
Up to the last years, hyaluronic acid has been employed as sodium salt in the theraphyxe2x80x94mainly in the ophthalmology, dermatology, surgery, articular therapyxe2x80x94and cosmetics. The salts of hyaluronic acid formed with alkaline, alkaline earth, magnesium, aluminium, ammonium and substituted ammonium ions may serve as carriers for promoting the absorption of drugs (see the Belgian patent specification No. 904,547). Heavy metal salts of hyaluronic acid, among these the silver salt, are employed as fungicides whereas the gold salt is employed for the treatment of arthritis (see the patent specification No. WO 87/05517). However, it is known the severe adverse effects of silver and gold compounds, i.e. their effects on the immune system, haematopoietic organs and nervous system [M. Shinogi, S. Maeizumi: xe2x80x9cEffect of preinduction of metallothionein on tissue distribution of silver and hepatic lipid peroxidationxe2x80x9d, Biol. Pharm Bull. (Japan) 16, 372-374 (1993); C. Masson et al: Rev. Med. Interne (France) 13, 225-232 (1992)].
We have surprisingly found, that hyaluronic acid associates, i.e. zinc and cobalt hyaluronates, which are useful for accelerating the epithelization of epithelium-deficient body surfaces, for healing crural ulcer as well as decubital ulcer, possess significant antimicrobial, more particularly antibacterial and fungicidal effects; in addition, within the antibacterial activity they proved to be active also against Helicobacter pylory bacteria recently considered to be responsible for the development of gastric and duodenal ulcers.
The antibacterial effects of hyaluronic acid associates are thought to be surprising since it can be supposed that the wound-healing epithelization-promoting effects of hyaluronic acid associates known until now are based on the above-discussed body-friendly behavior of hyaluronic acid during the wound-healing but the antimicrobial effect of hyaluronic acid associates cannot be concluded therefrom.
According to our investigations, zinc and cobalt associates of hyaluronic acid proved to be very active against either aerobic or anaerobic bacteria such as Staphylococcus aureus, Streptococcus sp., Pseudomonas aeruginosa, Salmonella sp., E. coli and Helicobacter pylon. 
Since Staphytococcus aureus and Pseudomonas aeruginosa are the two types of bacteria that are implicated in eye infections, the fact, that the hyaluronate complexes are effective against both of these microorganisms is very important and using the hyaluronic acid associates in the ophthalmology seems especially promising.
The intraocular use of sodium hyaluronate in the ophthalmology is known. Since 1980 it has been widely used in the case of cataracta surgery, artificial lens implantations, keratoplasty. When administered into the anterior chamber, it inhibits the collapse thereof during operation and protects the sensitive tissues and cells. After the surgical intervention, sodium hyaluronate is washed out from the eye to avoid the increase of ocular pressure occurring in some cases.
Sodium hyaluronate is used also in the treatment of the xe2x80x9cdry eyexe2x80x9d syndrome [F. M. Polack et al: xe2x80x9cThe treatment of dry eyes with Na-hyaluronatexe2x80x9d Cornea, 133-136 (1982)].
The topical antibacterial treatment of eye infections is an other important field of utilization of associates of the present invention.
Due to their antibacterial effect, zinc and cobalt hyaluronate associates seem to be particularly useful in the ophthalmological therapeutics; mainly zinc hyaluronate appears to be very promising in this area. In addition to its lubricant effect, zinc hyaluronate can advantageously be employed as a topical antibacterial agent in the ophthalmology; but it can be used also in the ocular surgery as a competitor of sodium hyaluronate widely used intraocularly since 1980, due to its antibacterial effect, to overcome the occurring mild inflammatory reactions [K. L. Goa et al: xe2x80x9cHyaluronic Acid, a Review of its Pharmacology and Usexe2x80x9d, Drugs 47, 536-566 (1994)] and to reduce the risk of infection during the operation. The role of zinc as an essential trace element in the eye has long. been recognized [D. A. Newsome, R. J. Rothman: xe2x80x9cZinc uptake in vitro by human retinal pigment epitheliumxe2x80x9d, Invest. Ophthalmol. Vis. Sci. 28, 795-799 (1987)]. Zinc hyaluronate has the particular advantage that, by using it, the decrease of zinc possibly occurring in eye operations can be avoided. Namely, hyaluronic acid as a polyanion can take away cations by the washing-out after operation. It cannot be excluded that zinc being bound also through a coordinative bond to hyaluronate, may be decreased in the eye. This phenomenon can be eliminated by using zinc hyaluronate.
It is known, that bacterial contamination of the eye is a serious health problem. The most frequent contaminant is the Staphylococcus group. Less frequently the contaminant is Pseudomonas aeruginosa which is also very dangerous to the eye. Infection of the eye with these bacteria can result in complete blindness over a 24 to 48 hour period. The infections of eye are frequently caused by contaminated ophthalmological solutions (eye drops, solutions for the storage of contact lenses or the like). Such contaminated ophthalmological solutions occur in consulting rooms, clinics and very frequently in the home use. The contaminating microbe most frequently belongs to the Staphylococcus group; however, the less frequent but more dangerous Pseudomonas aeruginosa is also capable of rapid growth in ophthalmological solutions (Remington""s Ophthalmic Preservatives, Chapter 86, 1588). Thus, the use of zinc and cobalt hyaluronate associates in the ophthalmology may be very important by their strong activity against both species of the above microorganisms.
The antimicrobial effect of the compounds was verified by microbiological investigations. Zinc hyaluronate and cobalt hyaluronate solutions according to the Hungarian patent specification No. 203,372 were used for these examinations whereas sodium hyaluronate solutions served as reference solutions.
The following Example 1 shows preparation of a 0.5% zinc hyaluronate solution. Unless otherwise noted the percentages hereinafter are always weight/volume values.
Solutions of 0.1 or 0.2% concentration respectively were obtained by diluting 0.5% zinc hyaluronate solution with distilled water of quality according to the example.